We propose to characterize regulatory and control processes in Na-K ATPase at the functional and physical levels, in order to understand better the role of this enzyme in the function of excitable tissues, especially the heart. Our preliminary results indicate that Na-K ATPase activity can be controlled by alterations in the affinity of the enzyme for monovalent cations, produced by pH and Ca. We intend to demonstrate that these alterations arise from conformational perturbations induced in the enzyme by binding of regulatory ligands at sites allosteric to ATP, Na, and K binding sites. Continuing our kinetic studies, we will characterize the effects of divalent cations at different pH values on affinities for Na, K, and ATP. We will also assay effects of metabolites and ions, e.g. ADP, HCO3 minus, and pharmacological agents, e.g. ouabain and related cardiac glycosides, diphenylhydantoin, and local anesthetics of the procaine family, on the basis of their possible roles in maintenance of membrane excitability or cardiac contractility. Conformational changes in the enzyme associated with alterations in Na or K affinity will be studied by circular dichroism of a highly purified ATPase preparation which has not been subjected to treatments that might alter lipid-protein interactions. Reactivity of amino acid side chains, especially sulfhydryl groups, will also be used as a probe of conformational changes, under conditions in which reactivity can be measured on enzyme which is initiating substrate turnover. Alterations in the fluorescence spectra of ethynyladenosine substrate analogues provide a third probe of conformational changes associated with regulation of ion affinities. We expect our results will demonstrate that Na-K ATPase regulation can be achieved not only by altering catalytic constants but also by changing the so-called E1 and E2 conformations of the enzyme associated with the sequence of steps in hydrolysis so that binding of monovalent cations is affected. These studies should aid in understanding the mechanisms of maintenance of ionic homeostasis in excitable tissues and regulation in membrane proteins. Bibliographic references: Josephson, Lee, Mangold, John, and Simon, Sanford. "An Automated Assay for Na-K ATPase Kinetics." Anal. Biochem. 60, 312-318 (1974).